Our long-term goal is to understand the molecular mechanisms of dentin biomineralization. The most abundant and important molecules in dentin are type I collagen and dentin sialophosphoprotein (DSPP). DSPP is a multifunctional protein that is cleaved into component parts by proteases. Proteases act by altering the properties of the proteins they cleave: by activating them, by allowing subunits to separate and function in different locations, and by degrading or inactivating them. Proteolysis is central to the biological activity of DSPP, and tailors its activities to serve the various requirements of intertubular and peritubular dentin formation and the maturation of dentin. Specific Aim 1: To identify the protease that catalyzes the initial cleavage of DSPP (generating DPP) and to determine if early cleavage products (DSP, DGP, and DPP) segregate preferentially into predentin, peritubular dentin, or intertubular dentin. The protease that releases DPP is assayed during purification procedures using a fluorescent peptide and confirmed by characterizing its cleavage of recombinant DSPP. Region specific DSPP antibodies are used in light and electron microscopy to immunolocalize each structural/functional domain. Special procedures are used to separate predentin and peritubular dentin from whole dentin, and DSPP-derived proteins in each extract are characterized. Specific Aim 2: To determine which DSPP-derived proteins are structural (long-lived) and which are transient (degraded) and characterize the enzymatic processes that determine this. Proteins are extracted at four successive stages of crown development and DSPP cleavage products in each stage are characterized. N-terminal sequencing identifies the specific cleavage sites catalyzed by enzymes in vivo. In vitro digestions identify the proteases that catalyze these cleavages. Specific Aim 3: To characterize the structural/functional properties of the major early DSPP- derived proteins and their long-lived cleavage products. DSPP proteins are tested for their ability to promote cell attachment and induce relevant changes in gene expression, and to bind collagen. The basis for DPP structural diversity is also determined. This research will advance our understanding of how proteases activate DSPP to promote predentin, peritubular and intertubular dentin formation, and degrade DSPP to facilitate dentin maturation. Understanding how dentin forms and hardens will help us induce the biological repair and regeneration of teeth and improve the diagnosis and treatment of dentin pathologies, which can arise from genetic or environmental factors, injury and disease. Defects in the dentin sialophosphoprotein (DSPP) gene cause dentinogenesis imperfecta and dentin dysplasia. We investigate how proteases cleave DSPP to support peritubular and intertubular dentin formation and degrade DSPP to promote hardening of formed dentin. A better understanding of dentin biomineralization is needed to develop treatments that induce the repair and regeneration of teeth.